This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The principle mechanism responsible for the contraction and relaxation of smooth muscle involves the reversible phosphorylation of the 20 kDa regulatory myosin light chain (rMLC). Typically, elevations in intracellular Ca2+ result in rMLC phosphorylation at the serine-19 (or adjacent threonine-18) residue by the Ca2+/calmodulin-activated MLC kinase (MLCK). The studies in this collaboration are intended to extend findings with respect to the physiological importance of this alternate-site phosphorylation in vascular smooth muscle, and the signaling pathways involved. Continued studies are proposed using accurate mass-driven analysis and rapid parallel MS/MS acquisition to further identify and clarify the physiological conditions under which this alternate site rMLC phosphorylation occurs.